Substrates for isolating, reacting and microscopically analyzing materials

ABSTRACT

An immobilizing device for biological material comprises a rigid support ( 12 ) carrying a substrate layer ( 20, 20 ′) of polymer having biological immobilizing properties, e.g. for amino and nucleic acids. Substantially solid ultra-thin substrate layers ( 20 ′) having a thickness less than about 5 micron, preferably between about 0.1 and 0.5 micron, and micro-porous, ultra-thin substrate layers ( 20 ′) having a thickness less than about 5 micron, preferably less than 3 micron, 2 or 1 micron are shown, which may be segmented by isolating moats M. The substrate layer is on a microscope slide ( 302 ), round disc ( 122 ), bio-cassette, at the bottom of a well of a multiwell plate, and as a coating inside a tube. Fluorescence or luminescence intensity and geometric calibration spots ( 420 ) are shown. Reading is enhanced by the intensity calibration spots. ( 420 ) to enable normalization of readings under uneven illumination conditions, as when reading by dark field, side illumination mode. The reference spots are shown being printed simultaneously with printing an array of biological spots or with the same equipment. Methods of forming layers of the device include controlled drawing from a bath of coating composition and drying, and spinning of C-D shaped substrates. Post-forming treatment is shown by corona treatment and radiation. Adherent metal oxides ( 14 ), silica-based materials and other materials are used to unite layers of the composite. In multi-well plates the oxide promotes joining of a bottom plate ( 95, 95 ′) and upper, well-defining structure ( 94 ) of dissimilar material. The oxides ( 14 ) also provide beneficial opacity to prevent light entering the glass support, for applying potential to the substrate, etc.

TECHNICAL FIELD

This invention pertains to substrates for isolating, reacting andmicroscopically analyzing bio-materials, especially proteins, andgenetic materials. The invention also pertains to methods for making andtesting the substrates, to bio-array products employing the substrates,and to methods of binding, reacting, assaying and imaging materials onthe substrates.

Embodiments of the invention in particular pertain to coated glassslides, multi-well plates and similar rigid supports that receive theprotein or genetic material and retain the material in precise positionwhile an assay is performed, the array is washed, and the altered arrayis analyzed for fluorescent emission. The invention also pertains tomulti-well plate constructions as well as to tubes, bio-cassettes,disk-form substrates and other configurations.

Embodiments of the invention pertain to techniques for isolating,binding and discriminating between different bio-molecules and forestablishing conditions for reaction.

Embodiments of the invention pertain to examination of tissue, forinstance biopsies of potentially malignant tissue or of contaminatedmaterials such as potable water, e.g. to enable detection of rareevents.

BACKGROUND

The search for improving and extending the capabilities of opticalanalysis have long involved considerations of the substrate on which thespecimen is supported during the analysis.

In the case of biological material, use has been made of polymericsubstrates, in particular, porous substrates also referred to as“membranes” and “matrices,” to immobilize the material while thematerial undergoes genetic analysis or is used for cell or proteinresearch. Historically, porous matrices were first created as filters,to separate particulates contained within a liquid. In the process, anumber of porous polymeric matrices were identified to have strongbinding affinity for a number of bio-polymers. These matrices became thesubstrates of choice for cytochemistry and bio-polymer studies,especially where radioactive labels were employed.

The ability of nitrocellulose membranes (also referred to as “cellulosenitrate”) to serve as substrates to bind single stranded DNA, i.e., toimmobilize DNA, was demonstrated by Nirenberg in 1965 in flow-throughassays. Such membranes were commonly formed using fibrous cellulose as astarting material.

Cellulose, to which nitrocellulose is related, is formed as a chain ofglucose units, which is the universal building material for livingcells. Nitrocellulose membranes benefit in this regard by relationshipto cellulose, and have been commonly used substrates because of theirmolecular binding properties. The membranes have been used to bindcells, bio-polymers, proteins, genetic material and nucleic acids, aswell as serving as substrates for non-biological chemicals.

The use of micro-porous polymeric membranes, in particular,nitrocellulose, for blotting bio-molecules from electrophoreticallyseparated molecules was developed by Southern for DNA-DNA interactions.The technique is commonly called “Southern” blotting in honor of thedeveloper. The other compass directions have been developed. “Western”blotting is a technique that has been employed to immobilize protein onan immobilizing substrate for protein-protein interactions.

Southern's need was for a method to identify the separated zones inelectrophoretic separation. The blotting method employed micro-porousnitrocellulose to specifically identify the electrophoreticallyseparated zones.

A brief outline of the original Southern blotting technique may helpunderstand the general function of the nitrocellulose substrate:

-   -   A sample, in this case containing DNA, is separated on a gel        media by electrophoresis and is denatured by treatment with        sodium hydroxide.    -   A micro-porous nitrocellulose membrane is placed over the gel.    -   Blotter paper is placed over the membrane to absorb the water        from the gel and a weight is added on top. The weight forces the        water and separated molecules into the micro-porous membrane as        the gel collapses beneath the membrane. This leaves an image on        the membrane comprised of the separated bio-molecules.    -   DNA from the zones is bound non-specifically to the micro-porous        nitrocellulose membrane.    -   The nitrocellulose membrane is washed and blocked by diffusional        methods.    -   For performing an assay, the nitrocellulose membrane is then        incubated with a solution of a known labeled DNA.    -   If the DNA added is an exact match to a zone of the DNA        immobilized from the electrophoretic separation, the labeled DNA        will bind and the zone will be labeled.    -   After successive washings, a visual image of the labeled zones        then is prepared using X ray film if a radioactive label were        used, thus identifying the zones.

Following the original development of Southem's techniques, in an effortto increase throughput, a trend developed to replace radioactive tracerswith fluorescent tags, with the stimulated fluorescent emissions beingimaged by optics. It was noticed, however, that available porouspolymeric membranes, themselves, exhibited fluorescent emission over awide spectral range. This fluorescent emission, as background noise,limited the use of polymeric membranes in fluorescent studies ofproteins. While nitrocellulose membranes have been identified as one ofthe least offenders, still, when used as a substrate material,nitrocellulose has been found to have objectionable fluorescence thathas limited both the accuracy of detection and the throughput of assays.

In the case of DNA, despite a continuing desire to employ polymericmembranes such as nitrocellulose, a way around the fluorescence problemwas found, by spotting arrays on glass or quartz slides, that haverelatively little background emission, using a layer of non-polymericsilane or GAP, and other such materials as adhesion promoters to whichthe biopolymer is directly bound. These adhesion promoter materials,despite their own significant problems, such as difficultly in obtaininga uniform thickness, noise contribution, and reactivity, have permittedsignificant success with small molecules. No similar technique hasexisted that is as effective for protein molecules, which areapproximately 1000 times larger than DNA. Resort, still, has often beenmade to membranes of considerable thickness of porous nitrocellulose orother self-fluorescing polymeric immobilizing material, the materialeither being self-supporting or backed by a support. In the case ofmicro-porous nitrocellulose on a backing such as a microscope slide,typically the nitrocellulose has been at least 10 micron in thickness,and its self-fluorescence has remained a limiting factor for assaythroughput. The significance to biology and to the clinician of the needto conduct higher throughput, large scale assays of protein arrays isdiscussed for instance in Chin et al., U.S. Pat. No. 6,197,599.

New insights are presented here about the substrates on which many ofthe known protein assays can be conducted. These insights lead broadlyto techniques that increase throughput and achieve higher accuracyimaging of fluorescently- or luminenscendy-labeled proteins and otherbio-materials for large scale assays for research and for clinicaldiagnosis.

Some Prior Techniques with Nitrocellulose

Referring specifically to the practice of depositing spots of biologicalfluid on a solid or micro-porous surface to create a microarray, thishas been widely described. In the case for instance of using glassslides bearing porous membranes of nitrocellulose, e.g. of 12 to 15micron thickness, the supplier, Schleicher & Schuell, has recommendedcreation of each spot of the biological fluid with as much as 50 nl offluid suspension, or more.

As a different nitrocellulose approach, using less suspension, spotscomposed of smaller amounts of liquid mixture of bio-molecules withnitrocellulose in a colloidal form or otherwise, have been formed on aglass or other support, where the nitrocellulose is either dissolved orin suspension in a common solvent. Additional solvents are introduced tocause desiccation of the deposit, resulting in a porous matrix thatserves as an immobilizing structure for the intermixed biologicalmaterial. This technique has been described by Pinkel in NatureGenetics, Volume 20, October, 1998, and by Audeh et al., U.S. PatentApplication Publication 2002/0015958. These publications, involving verythin deposits or spots of the mixture of the bio-material withnitrocellulose on a support, have so far failed to advance the state ofthe practical art. These processes appear to have an inherent source ofuncertainty or error, as they do not permit spot-to-spot evaluation ofthe contribution of the nitrocellulose or other immobilizing substratematerial to the signal detected from the spots of fluorescently labeledproteins. In the technique, the fluorescent signal from a spot itself isnecessarily the sum of the emission of the glass or other support, theporous nitrocellulose or other immobilizing material and the biologicalmaterial itself. As these emissions are all combined, no simple methodof separation exists for the signal from the biological material fromsuch spots of biological/nitrocellulose material.

In more common assays with the available much thicker but continuousmembranes of nitrocellulose mentioned, and with other immobilizingpolymers, subtraction of perturbation noise is commonly used. Owing tothe general uniformity of the thickness of such substrates across thesupports, standardized software can measure the emitted noise signalfrom the unspotted membrane and automatically subtract a valueapproximately representing its noise signal from the total signalderived from the spot In the case of the spotted Pinkel or Audeh et al.mixtures, the local vicinity beyond the spot does not contain acontinuation of the substrate material that contributed to thefluorescent background at the spot, so the subtraction technique cannotbe used to remove the effect of the substrate material. The magnitude offluorescent energy as measured by the level of signal detected by aconfocal scanning microscope such as the Affymetrix 428 Scanner hasshown that a substantial error in the measurement of sample fluorescencecan be introduced by the Pinkel or Audeh et al. process, and nosuccessful variation of the technique that allows some form ofsubtraction has yet been found.

Ultra-Thin, Low-Noise Immobilizing Substrates

A new and different approach is presented to immobilizing and imagingfluorescently labeled biological materials. It employs a continuous,ultra-thin layer of a substrate of polymer that has biological bindingproperties, for instance, nitrocellulose. The technique is effective,using protein-immobilizing polymeric substrates, to enable imaging offluorescently labeled proteins. The technique has other potentialwidespread uses, such as with proteins labeled with luminescent tags,and with other bio-materials labeled with fluorescent or luminescenttags. The technique may be used to advantage with viruses, peptides,antibodies, receptors, and other proteins; with a wide range of otherlabeled biological materials including plant, animal, human, fungal andbacteria cells; with nucleic acids, as a very practical substrate, withfewer problems than other materials, e.g. for cDNA clones, DNA probes,oligonucleotides including synthetic oligonucleotides and polymerasechain reaction (PCR) products; and with labeled chemicals as well. Ithas the attractive potential of being a low-cost, practical substrate ofchoice over the prior materials, such as GAP for instance, (GAP, becauseof high reactivity, requires costly and time-consuming precautions toavoid contamination.)

It has been found that a superior immobilizing substrate, suitable forreceiving deposit of an array of spots of bio-polymers, is provided by acontinuous ultra-thin layer of polymeric substrate material havingbiological binding properties, i.e., (a) in a 3-dimensional micro-porousform, an ultra-thin layer of thickness of less than 5 micron, down toless than a micron in thickness or approximately equal to the size ofthe pores, with pore size respectively from about 3 micron to ½ micron,or (b) as a solid surface coating, of a thickness less than 5 micron,less than 3 micron or thinner, down desirably to submicron thicknesses,e.g. 0.1 to 0.5 micron, or even as a molecular layer.

Polymer substrates of these dimensions are found useful to immobilizeproteins and the broader categories of materials mentioned. Further, itis found that such continuous, ultra-thin polymer layers can readily beformed.

The uniform ultra-thin layer of biological-material-immobilizing polymeron a rigid substrate has been found to be capable of enduring theconditions of printing of spots of bio-polymers in precisely knownpositions, of conducting the assay, of application of successive washes,and, following handling, of being microscopically analyzed by stimulatedemission. The ultra-thin layer is found to significantly reducebackground noise attributable to parasitic fluorescence of theimmobilizing material and to otherwise offer advantages due toconsiderations that will be described.

It is found also that a continuous ultra-thin layer can have suchuniformity that it enables its signal contribution from its area lyingbeyond the spotted material to be subtracted from the measurement of thespots in a reliable manner, further increasing the quality of signalsfrom that obtainable by prior techniques.

Those skilled with respect to protein-immobilizing membranes may havesupposed that a significant depth of porous nitrocellulose or otherprotein-immobilizing substrate, i.e., 10 micron or more in availablecommercial products, would be important. Those skilled may have supposedthat the significant depth of present commercial membranes was requiredto enable forming a uniform and durable membrane that could surviveprinting of spots, conducting the assay, applying successive washes andhandling the unit through analysis, while still holding the depositedspots reliably in their precisely known places. Or, those skilled mayhave believed that current commercial thicknesses of the substrate wererequired to enable reliable manufacture, or to provide aliquid-receiving volume below the deposit sites to enable the carrierliquid to drain downwardly. Or, those skilled in the art may havebelieved a significant thickness of the immobilizing substrate wasrequired, to provide a thickness-to-variance-in-thickness ratiosufficient to enable a reliable subtraction technique for correcting forauto-fluorescence, etc.

It has been found that no such requirements are in fact necessary. Ithas been found that durable, ultra-thin continuous substrates of polymerhaving biological binding properties can be readily fabricated of lessthan about 5 micron thickness, and that spot formation and precision oflocation is not adversely affected by the steps of spotting, assaying,washing, handling and analysis.

It has been found that, using coating techniques that are conventionalfor thin coatings in other contexts, the inherent variation in thicknessof the polymer coating is sufficiently small, relative even to the smalloverall thickness of the ultra-thin substrate, that a signal from theadjoining unspotted area of the continuous coating may be used in thedescribed subtractive techniques to enable acquisition of superiormicroscopy results.

It has been found that a solid (non-porous) film of 3 micron, down tothickness under 75 nm, even down to molecular thicknesses, ofimmobilizing polymer substrates, and in particular, of nitrocellulose,can be formed and successfully employed in high throughput protein andother bio-material assays.

It has also been realized that such ultra-thin substrates may be alteredafter forming as a coating or substrate, as by corona discharge, atomicparticle or radiation bombardment or by controlled energy excimer laserbeam treatment, to improve binding and immobilization topology orconditions.

In making such developments, the importance has been recognized of thefact that bio-molecules bind to the surface of the nitrocellulose orother immobilizing polymeric material, while parasitic fluorescence isemitted from the entire volume or bulk of the material illuminated bythe inspection technique. When microarrays have been spotted oncommercial membranes (10 micrometer or thicker membranes), thebiological material normally accumulates at the outer portion of thethickness of a membrane supported on rigid non-porous support, e.g., ononly 30% or 40% of the total thickness of the membrane. This is due tothe fact that flow-through conditions for the bio-molecules areobstructed when a very small volume of volatile fluid supporting thebiological matter is deposited. Bio-polymers and the carrying fluids wetthe surface, the necessary phenomenon, and saturate the pores to ashallow depth and block further penetration as the liquid carrierseparates, migrates beyond or evaporates.

The importance has been realized of the fact that polymericbio-immobilizing materials such as nitrocellulose behave in anapproximately linear manner and emit fluorescent radiation inrelationship to the volume of material exposed to the excitation beamand the level of excitation and that a layer of the material ofthickness limited to less than 5 micron and preferably less than 3micron thick and in important cases even less than 1 micron thick canprovide significant advantage.

In the case of ultra-thin micro-porous substrates provided here, thepercentage of the volume of porous material that actually bears thebiological material, relative to the total volume of the substratematerial presented to the collecting optics, may be greater than 50% andadvantageously in many case, greater than 75%, unnecessary volume of thematerial and its deleterious fluorescence being avoided.

Experimentally it has been determined that the parasitic emission offluorescent light increases with the thickness of a micro-porousmembrane with thickness of 1, 2, 3, 4, 7 and 14 micron.

It has further been realized that the parasitic fluorescence emission ofa porous membrane of a given amount of nitrocellulose or otherbiology-binding polymeric material per unit area of the supportstructure can be many times greater (measured in one case to beapproximately 6.4 times greater) than that of the same materialpresented as a transparent solid film. The nitrocellulose in itstranslucent/semi-opaque 3-dimensional porous membrane form is observedto absorb excitation radiation to a much greater degree than the samematerial in transparent semi-crystalline form. In addition, a relativelythicker porous membrane also reflects or scatters some of the excitationenergy to a much greater degree than a thinner and especially,transparent, membrane.

Furthermore, it has been observed that an ultra-thin transparent solidmembrane of polymer having biology binding properties, in reflecting aminimum amount of excitation energy, minimizes the exclusion requirementof the filter that is required in a collecting system to separate thefluorescent emission energy to be detected from the excitation energy.

It has also been recognized that the strength of the fluorescentradiation signal emitted toward the collecting optics by thefluorophore-tagged bio-polymers bound to the immobilizing polymericmaterial is not only a function of the quantity of the bio-polymerpresent and of the energy of the excitation source. It is also afunction of the location of the emitting bio-molecules with respect tothe top surface of the immobilizing medium. Bio-molecules may be locatedon the outer surface or buried to varying depths of a 3-dimensionalmembrane structure. Fluorophores attached to the molecules locatedwithin a 3-dimensional matrix below the outer surface are twicehandicapped when compared to similar molecules on the outer surface. Theenergy intensity penetrating a semi-opaque, diffusive material, such asa highly porous polymeric material, decreases in function with thedistance traveled and the absorption characteristics of the medium. In asimilar manner, the stimulated fluorescent light from excited moleculesburied within the matrix is absorbed to some degree or scattered beforeexiting to be collected by the optical system. The deeper a particularfluorophore is in a 3-dimensional porous polymeric structure, to somedegree, the less intense will be its fluorescent emission at thecollecting optics.

Accordingly, the novel, solid ultra-thin polymer film of immobilizingnitrocellulose or other bio-material-immobilizing polymer material isseen to be of considerable importance. It is recognized that a surfaceof solid nitrocellulose or other solid immobilizing polymer may usefullyprovide sufficient binding sites (for bio-polymers, cells or smallfragments of tissue or other material to attach to), on a single plane,in a deposited spot of useful size for an array to be assayed. A numberof binding sites equal to that of a surface folded in a small pore, 3dimensional structure (such as that of a micro-porous polymer membrane)is obtainable with a solid coating by increased spot size. It isrecognized that binding sites on a general plane in some ways offersbetter binding opportunity, e.g. equal opportunity for attachment ofbio-polymer molecules to all sites, than is possible within anequivalent surface tightly folded in a 3-dimensional form. This isespecially true in comparison to micro-porous polymer structures in thecase where pore size may vary, and in cases where bio-deposits aredependent upon concentration, drying conditions, etc. of the spottedfluid. Especially for large protein molecules, this consideration isbelieved to be obtainable from assay to assay by performing assays ofthe proteins or other bio-polymers on solid, or modified solidultra-thin coatings of immobilizing polymer material.

Continuous ultra-thin micro-porous polymer substrates, and solidsubstrates of bio-material-immobilizing polymer material, supported onglass, metal or plastic, used to immobilize fluorescently-tagged orluminenscently tagged bio-polymers, can achieve superior signal-to-noiseratio and other advantages that provide superior information ordiagnostic efficacy.

Excellent analysis results have been obtained employing nitrocelluloseas the ultra-thin micro-porous polymer material or as an ultra-thinsolid polymer coating, while very desirable results are also realized tobe obtainable with polystyrene. Other ultra-thin polymers that havebiological binding properties may also be used e.g., cellulose acetate,cellulose triacetate, ethyl cellulose, activated nylon,polytetrafluoroethylene (PTFE), polyvinyl difluoride (PVDF), polyamides,polyvinylchloride, di-vinyl benzene and agarose, including copolymersand blends.

In one specific embodiment, a continuous micro-porous polymer matrixthinner than about 5 micron, and preferably as thin as 3, 2 or 1 micronor less, is provided to support bio-polymers under study. With the useof this structure, while the parasitic noise is reduced according to thethickness of the ultra-thin, polymeric substrate, it is found that thefluorescing signal is minimally reduced in comparison to use of thepresently available commercial materials, the transferred volume offluid not being appreciably altered in its course into the thickness ofthe ultra-thin material. Ordinarily the 3-dimensional matrix offers alarger binding surface (more bio-material binding sites) than thefootprint of the same support.

Another important embodiment, however, is the ultra-thin, solid, i.e.non-porous, coating of polymer with biological binding properties,thinner than 5 micron, preferably less than 3 micron and preferably asthin as 2, 1, 0.5 or 0.1 micron, or even at molecular thicknesses,deposited on a rigid supporting medium of extremely low fluorescentproperties, such as low fluorescence glass, fused quartz, ceramic, PMMA,polystyrene, other plastic or metal. The overall background-perturbingeffect of such a bio-compatible substrate is preferably of the order ofor even less than that of its supporting rigid structure.

In these embodiments, the number of photons necessary to obtain astatistically reliable signals dictates the spot area dedicated forattachment of the bio-polymers. Preferably, this area is approximately aspot with diameter greater than 100 micron in diameter and less than1,000 micron, preferably less than 500 micron. In many cases, thepreferred spot sizes of the bio-polymers are below about 500 micron, forinstance 100 to 400 micron, 150 micron and 300 micron being common dotsizes. This enables the formation of suitable microarrays with provisionfor the needed sequence of dilutions and provision of process referencespots to enable large scale, high speed throughput of the assay andanalysis. The system is more economical, with respect to amount ofbiological material required per spot, e.g. less than one nl per spotrequired, in comparison to prior art schemes of spotting on 10 or 12micron or greater thickness micro-porous nitrocellulose on glass, forwhich a recommended amount by one supplier for an individual spot hasbeen as high as 50 nl or higher.

Methods of manufacturing such ultra-thin, immobilizing polymer layersare provided that are found to produce particularly good substrates.

Novel methods are provided of depositing ultra-thin coatings ofnitrocellulose or other immobilizing polymer, i.e. material havingbiological binding properties, on a suitable solid support, in manycases, a microscope glass slide having approximate dimensions of 25×75mm, by about 1 mm thick, as will now be described.

Blank glass microscope slides are obtained, such as part No. 2951 fromErie Scientific Co. in Portland, N.H., with a short frosted section atone end. These are pretreated by applying a surface adhesion promoterand/or a layer permitting the application of indicia to the slide forimportant purposes such as identification and serialization,registration for microscopic analysis or other processes, provisionalclassification markings, or simply for presentation or branding. Anumber of choices can be made. Two preferred embodiments are:

-   -   (a) A painted/covered region is applied over the frosted area        with or without the addition of a 1 to 3 mm (and preferably        2 mm) wide frame tracking the outer periphery of the slide. The        painted region over the frosted area may later be laser-marked        with identification and serialization or other marking as        desired. The process used may impart non-serialized markings.    -   (b) A coating is applied over the entire slide on the frosted        surface side by vapor deposition or sputter coating e.g.        tantalum followed by air oxidation to form a thin layer of        tantalum oxide in order to provide opacity ranging between 10%        and 90% with respect to a nominal laser wavelength of 635 nm.        The coated region over the frosted area is suitable later to be        laser marked with identification and serialization or other        marking including non-serialized markings. Also the region to be        spotted may be divided into sub regions (or islands), e.g. by        removing a circular or square moat of tantalum oxide surrounding        each individual array. Such isolated region may serve to protect        the spotted array portion from delaminating when an adhesive        gasket is applied outside of the moat during experiments.

In an alternative manner laser marking and segmentation may be performedfollowing coating.

Preferably a laser will be used with wavelength absorbed by the coatingto be removed and not absorbed by the material of the support, glass orother.

Laser ablation of the coating over the frosted region may serialize theslides or add identification or registration markings for automaticoptical unit or information retrieval. Ablation enhances dataacquisition reliability in processes using a variety of equipmentincluding commercial bar code readers.

Advantageously, after application of an adhesion promoting layer, atleast one durable sensitivity calibration spot may be applied. Thesensitivity calibration marking is provided to act as a fiduciarymarking for geometrical reference, and by suitable choice of itsmaterial, serves as a standard fluorescence reference in order todetermine and accommodate long-term variations in opticalinstrumentation.

Similar fluorescing calibration spots are applied on the outer surfaceof the completed substrate. In advantageous cases they are applied in alow-density pattern interspersed with high density biological spots, andused for calibration, or for normalization in instances where unevenexcitation illumination may occur, e.g. when employing illumination atan angle to the normal to the plane of an array, as in imaging viadark-field reflectance mode.)

Preferably, the calibration compound is selected to have a broadfluorescence spectrum. A temporally stable material, such as polyimidepolymers (Kapton), exhibiting broad band, standard fluorescence, i.e.,yielding fluorescence at a wavelength in reliable manner, is selected asthe reference fluorophore and deposited on a slide surface with solventfollowed by solvent evaporation. Typical spot diameters may be 150micron and 300 micron, and can be applied using commercially availablebiology printers (sometimes referred to as “spotters” or “microarrayinginstruments”.) The precise amount of material deposited is unimportantsince polymers such as Kapton are optically opaque, and detected.fluorescent emission from the polymer occurs at or near the surface ofthe deposited material giving reproducible quantum yields.

The use of the calibration material applied to each slide allows forinstrument self-calibration, i.e., auto calibration, per slide. Adistribution of calibration spots, in number and spacing dependent onthe non-uniformity of excitation illumination incident on a slide, maybe employed. As few as six distributed in an array may suffice althoughlarger numbers also are employed, depending upon the characteristics ofthe reader system.

A preferred process for preparing the glass microscope slides includesthe removal of all particulates and most organic matter via mechanicalmeans using solvents and detergents. The slides are subsequently left todry in air. The active surface (as defined as the surface with frostedarea) is then subjected to ozone treatment, e.g. to remove residualorganic matter and enhance the adhesive properties of the surface. Theozone reactions may be activated using corona exposure or UVillumination. In a preferred embodiment, the ozone/corona treatment isinduced by translating the slide at a speed between 2 and 8 cm/min(preferably 4.4 cm/min) past a corona discharge while exposing surfaceto be treated normal and approximately between 1 and 4 cm (preferably 2cm) to the jet of a standard 2.5 cm. round head of a laboratory coronatreater (model BD-20AC from Electro-Technic Products Inc., Chicago,Ill.) operating near its optimal level. Preferably the pressure,temperature, and humidity are held within the human comfort zone of65°-72° F., one atmosphere, and humidity between 30 and 70%.

Alternately, such slides are cleaned of debris as well as of anybiological products such as by washing them for approximately 30 minutesin an ultrasonic bath with a detergent and subsequently holding them inan oven at about 450° C. for approximately 8 hours.

If the preferred embodiment of pretreatment (a) above is used, theslides are then coated with a less than 1μ thick layer with colloidalsilica or soluble silicate. For this purpose, LUDOX CL, LUDOX CL-X, orLUDOX TMA suspended in water is employed, available from Sigma-AldrichCo. An equivalent product may be obtained from other sources. For thispurpose, slides are held for 1 second to 1 hour in a bath of 1% to 10%(preferably 3.3%) colloidal silica and exhumed (drawn from the bath)preferably at a constant rate between 0.1 and 10 in/min (preferably 0.5in/min), along a path parallel to the plane of the microscope slide toform a coating (referred to as a “drawn coating”). This is followed bydrying in air. Preferably, the environmental pressure, temperature, andhumidity are held within the human comfort zone as previously described.In producing a final substrate layer upon which the biological materialis to be immobilized, the slides are preferably submerged in a solutionof desired substrate material and withdrawn from the solution at anappropriate uniform rate, to form a drawn substrate layer. This coatingprocedure is performed under defined environmental conditions and usinga solution adapted to such standardized conditions.

The final coating fluid may be selected from a number of possibilities.Three preferred embodiments employ compositions that form polystyrenefilms, nitrocellulose films, and nitrocellulose microporous membranes.The coating solutions can be employed over a wide range ofconcentrations. Advantageously the solutions can be normalized to agiven set of operating conditions for production advantages. In theexamples to be given, the three compositions of nitrocellulose andpolystyrene are normalized to a set temperature of the still environmentand a set draw rate, specifically 26° C. and 1 in/min, respectively, forproducing films of 0.1μ thickness of polystyrene and nitrocellulose andan adherent porous membrane of less than 5μ of nitrocellulose.

In a specific example, polystyrene film is produced by dissolving 5 gmof polystyrene pellets from Dow (pre-dried between 90° and 100° C. for24 hours) in 100 mL of amyl acetate with low shear mixing. Low shearmixing is obtained by rolling the ingredients in a glass vessel at 4rpm. Slides are submerged in a vat held at 26.0°±0.5° C. containing thecomposition and subsequently withdrawn at a uniform rate between 0.1 and10 in/min, preferably 1 in/min. Optimally, the process is performed in a5 cubic feet hood with a controlled atmosphere of 33% humidity and 26°C. with draw rate matched to the composition and the controlled processconditions. Following slide withdrawal, the processed slides are kept inthe hood for 1 to 5 minutes. To reach a desired thickness of the coatedslide, more than one dip may be employed.

In an embodiment for producing nitrocellulose films, 1.33 gm ofnitrocellulose and 0.1 gm of dehydrated tin (II) chloride (SnCl₂) aredissolved in 100 mL of amyl acetate. Slides are dipped into (submergedin) a vat held at 26°±0.5° C. containing the composition and withdrawnat a uniform rate between 0.1 and 10 in/min, preferably 1 in/min.Optimally, the process is performed in a 5 cubic feet hood with acontrolled atmosphere of 33% humidity and 26° C. Following slidewithdrawal, the processed slides are kept in the hood for1 to 5 minutes.To reach a desired thickness of the coated slide, more than one dip maybe employed.

In an embodiment to produce nitrocellulose microporous membranes, 4.14gmof nitrocellulose and 0.1 gm of dehydrated tin (II) chloride (SnCl₂) aredissolved in 55.6 mL of methyl acetate, 26.3 mL of ethyl alcohol, and13.6 mL of butyl alcohol, 2.94 mL of water, and 1.11 ml of glycerol.Slides are dipped (submerged in) into a vat held 26.0°±0.5° C.containing the composition and subsequently are withdrawn at a uniformrate between 0.1 and 10 in/min, preferably 1 in/min. Optimally, theprocess is performed in a 5 cubic feet hood with a controlled atmosphereof 33% humidity and 26° C. Following slide withdrawal, the slides arekept in the hood for 2 to 5 minutes. To reach a desired thickness of thecoated slide, more than one dip (i.e. immersion) may be employed.

In other embodiments for each type of coating, tin (II) chloride (SnCl₂)may be omitted, and other solvents may be substituted for the solventslisted above. Acetone, DMSO, or ethyl acetate are commonly used asalternate solvents for nitrocellulose and polystyrene.

In preferred embodiments, slides prepared as described above are surfacetreated to enhance the their binding capacity to biological material. Ina preferred embodiment, corona treatment is induced by translating theslide at a speed between 2 and 8 cm/min (preferably 4.3 cm/min) whileexposing the surface to be treated normal to the jet of a standard 2.5cm. round head laboratory corona treater at a distance of approximatelybetween 1 and 4 cm. The laboratory corona treater (model BD-20AC fromElectro-Technic Products Inc., Chicago, Ill.) should be operated nearits optimal level. Preferably the environmental pressure, temperature,and humidity are held within the comfort zone as described previously.

Possible contaminants (coatings or deposits) on the backside of theslide that may become loose in further processing must be removed. Thebackside of the slide may be cleaned during or after dipping and pullingfrom the vat or prior to packaging. Preferably, the finished slides arestored in dry nitrogen.

If a solid, ultra-thin, immobilizing polymeric film is desired, it isdesirable to ensure that no liquid miscible with the solvent such asAmyl Acetate and having slower evaporation properties is present.

If a porous or 3-dimensional ultra-thin membrane is desired, water oranother common porogen is added to the Amyl Acetate or other solvent toproduce the pores. This is a technique known to those knowledgeable inthe art.

Various solvent/nitrocellulose concentrations, etc. are listed inChapter 5, pp. 116-157 of “Synthetic Polymeric Membranes” by RobertKesting—McGraw-Hill, 1971, the book hereby incorporated by reference inits entirety. For polystyrene, preferred solvents are methylene chlorideand acetone. For polystyrene, as well as PVDF and many of the otherimmobilizing polymer substrates, other suitable solvents aredimethylformamide (DMF) and dimethylsulfoxide (DMSO). Chloroform andother solvents recognized for the respective materials, as determinedfrom readily available references, may be used.

In other embodiments, other techniques are employed to mass-produce theultra-thin coatings of nitrocellulose or other bio-material-immobilizingpolymer material, as by Chemical Vapor Deposition (CVD).

While it is presently believed that ultra-thin layers of nitrocelluloseand polystyrene have particular benefit, and are per se novel asdescribed, also, as has been noted, other bio-material-immobilizingpolymers in molecular or colloidal form, can be formed in similar mannerto make ultra-thin, immobilizing polymer coatings on bio-chips. Typicalof such materials, as noted, are cellulose acetate, cellulosetriacetate, ethyl cellulose, activated nylon, polytetrafluoroethylene,polyvinyl difluoride (PVDF), polyamides, polyvinylchloride, di-vinylbenzene and agarose, including copolymers and blends.

It is further recognized that techniques that have been developed in theoptical industry, to produce elements having optical properties, may innovel manner be brought over to produce the ultra-thin polymeric,bio-material-immobilizing substrates themselves, such as by forming anultra-thin layer of solid polystyrene upon a glass microscope or slidesupport, e.g. to form a surface waveguide for exciting radiation.Importantly, techniques from the optical industry are also used in novelmanner to provide adhesive-promoting primer layers on a rigid support,such as metal or metal oxide coatings. Similarly such coatings haveother novel functions such as blocking passage of unwanted radiationbetween the substrate layer and its rigid support and applyingelectrical potential. These are formed prior to applying the ultra-thinpolymeric, bio-material-immobilizing layer, and as described below, arefound useful for other purposes.

Improvements in Forming Inspecting and Using Substrates

Beyond the issues of low fluorescence noise from substrates to whichbiological material is bound, new concepts are also presented here aboutsubstrates and their supports for isolating, bonding and reactingbio-molecules, and more generally for high accuracy optical analysis ofsmall samples and for detecting rare events in biological tissue andother materials.

It is realized that adhesion promoters, particularly, adherent metaloxides such as tantalum or aluminum oxide, soluble silicates such assodium silicate, and colloidal silica, (which have optical transmissive,absorptive and electrical properties developed for use in other fields)are useful for new and important biology analysis functions, accordingto other aspects of the invention. The functions relate both tomicroscopy and to the manipulation and reaction of bio-molecular andother bio-materials. They serve the important function of being unitedto adjacent layers, while providing new functionality in support ofbio-microscopy.

As previously indicated, according to an aspect of the invention,tantalum or aluminum oxide, a soluble silicate such as sodium silicate,or colloidal silica, as well as other similar adherent metal oxides,used as intermediate layers, enable union of inorganic supportmaterials, such as glass, fused silica, ceramic, silicon and metals suchas gold, aluminum, silver, on one hand, with, on the other hand, organicmaterials, including polymeric materials, capable of immobilizing orbeing otherwise compatible with bio-molecules, e.g. nitrocellulose,polystyrene, cellulose acetate, ethyl cellulose, activated nylon,polytetrafluoroethylene, polyvinyl difluoride (PVDF), polyamides,polyvinylchloride, di-vinyl benzene, agarose, and including copolymerand blends of such organic materials, as well as fluorescently stablereference polymers such as polyimides (Kapton). Likewise, an adherentlayer, e.g. one of the metal oxides, can be a compatibilzing an adherentmiddle layer between one class of organic material and another class oforganic material, each of which bonds better to the adherent layer thanto the other. An example is a polymeric material suitable to provide adimensionally stable structural support such as polycarbonate, and thelisted organic materials suitable for providing a deposit-receiving orimmobilizing surface for bio-molecules and other bio-materials,including cells and tissue fragments, etc. It is further found thatadherent metal oxides such as tantalum oxide can be deposited in a widerange of controlled thickness without appreciably affecting theiradhesion properties for such uses, and that important new functions canbe achieved with the material. Deposited thickness causing almostcomplete absorption and almost 90% of normal incident light from a redlaser at approximately 635 nm, have been found to exhibit reliableadhesion properties between glass and nitrocellulose film and othermembranes such as polystyrene film. Thus the material is useful in bothreflectance and transmission imaging modes

With tantalum or aluminum oxide, sodium silicates, or colloidal silicaor another adherence promoting layer that is appropriately transparentto the wavelength utilized, along with employing substrate and supportmaterials that are likewise transmissive, supports for biologicalmaterial are provided that enable excitation and inspection ofbiological or chemical reactions from different sides of the support,from opposite sides, i.e. in both reflectance and transmission modes, aswell as from the edges.

With a deposit of functionally opaque thickness of tantalum oxide orother adherent, light-absorbing material, supports for biologicalmaterial are constructed such that the inspection of biological orchemical reactions is observed from only one side, with the benefit ofblocking optical perturbation or polluting effects which may originatein the supporting substrate, such as fluorescence or luminescence. Byblocking most incident radiation from reaching the support, most of sucheffects are not stimulated in the support, and such effects, to theextent stimulated, are substantially blocked from being transmitted fromthe support to the associated vision instrumentation. Likewise straylight reaching the support or light intentionally applied is blockedfrom reaching the instrumentation.

This feature of the invention ties in with an additional aspect of theinvention, the modification of the surface of polymer substrate films,such as nitrocellulose or polystyrene, to enhance affinity or bindingproperties for the bio-material or molecules. The modification may be tochange the topology, the chemical nature or the charge state of thesurface. In advantageous cases, the substrate material is attached tothe rigid substrate via an adherent conductive layer such as thetantalum oxide and/or sodium silicates or colloidal silica. Surfacemodification conditions to enhance the binding affinity of the surfaceto biological molecules such as protein or DNA or any molecule ornatural or synthetic oligonucleotides can be corona treatment, flametreatment, or bombardment as with ions, electrons or atomic orsub-atomic particles, or radiation including gamma rays or X-rays.

Another aspect of the invention is the use of the conductive tantalumoxide layer or other adherent, conductive layer or coating to establishan electrically polarized outer surface on the substrate. A positiveelectrical charge potential established on the layer produces a similarpotential on the nearby substrate surface. This can attract to thesurface negatively charged particles or ions or molecules orpolynucleotides or artificially charged viruses or bacteria and/or canrepel positively charged, similar molecules or particles. Thiscapability is employed to select from similar molecules or particlesaccording to their electrical charge.

Also, since no electrostatic force acts upon molecules with neutralpotential, these are not attracted to or repelled from the surface. Thusa powerful sorting mechanism is now provided to attract and/or separatemolecules or particles according to their electrostatic charge.

Transparent, electrically conductive coatings (e.g. indium oxide) andsemi-conductive layers may be applied in the support sandwich fordesired effects, depending upon the application. Any of these featuresare advantageously incorporated in novel devices to support biologicalreactions, including:

-   -   Supports in the shape of microscope slides, typically planar of        approximately 25×75 mm width and length, 1 mm thickness.    -   Supports in the shape of bio-cassettes, e.g. those of various        common configurations.    -   Supports in the approximate form of CD disks.    -   The wells of multi-well plates.    -   Reaction tubes.

A further aspect of the invention relates to the bonding of a bottommember to the upper bottom-less structure of a micro-well plate. On abottom plate of glass (or organic material) a surface pattern oftantalum oxide or other adherence-promoting layer is applied. It may bein a pattern which matches the pattern of the lower edges of thebottom-less upper structure of the micro-well plate being assembled.This facilitates the manufacture of the composite well-plate, enablingthe upper structure to be of material (e.g. polystyrene) that isadhesively incompatible with the bottom plate (e.g. glass). The spacesbetween the pattern may be treated to carry ultra-thin immobilizinglayers as described.

In a related aspect of the invention, an adhesively incompatible supportplate, e.g. of glass, intended to form the bottom of a multi-well plateby bonding to the bottom-less upper structure, is coated with a film ofan adhesion promoter, e.g. the tantalum oxide and/or colloidal silica orsodium silicates discussed, followed by application of an upper film ofpolystyrene or other substance that is adhesively compatible with theupper structure, as well as being capable of performing as animmobilizing substrate. The bottom-less upper elements of the micro-wellplate may thus also be of polystyrene and joined to a uniformly coatedglass. The union may be enhanced by heat or as well as by the temporarypresence of a solvent such as Amyl Acetate or the temporary presence ofa solution of polystyrene in such a solvent.

The glass surface, coated with a thin film of polystyrene, may have itssurface adhesion enhanced as described above, e.g. by corona discharge,or rendered porous, to serve as, a bio-material receiving substrate atthe bottom of the well.

A uniform film of polystyrene coated over the adhesion promoter on theglass plate, may be an ultra-thin layer as previously described, and itsareas within the grid of the multi-well plate may thus be suitable forreceiving deposit of bio-molecules as previously discussed.

The desirable glass bottom micro-well plates can be reliablymanufactured and provided with the layers for the various featuresdescribed above in relation to glass support structures forimmobilizing, reacting, assaying and analyzing bio-material. Similarprocesses are applicable to other choices of plastic material for themicro-well plates.

Important aspects of the invention are summarized as follows.

A device for immobilizing biological material is provided comprising apolymer substrate layer having biological immobilizing properties,preferably for protein or nucleic acid, the substrate layer deposited ona rigid support, and having an outer deposit-receiving region exposed toreceive the biological material, wherein the substrate layer isultra-thin, having a thickness less than about 5 micron.

Preferred embodiments of this aspect of the invention have one or moreof the following features.

The substrate layer has binding properties for the biological material.

The rigid support defines a straight support surface e.g., a planarsurface such as that of a microscope slide or a cylindrical surface, andthe substrate layer is a drawn coating applied directly or indirectly tothe rigid support in the direction of the straight surface, preferablydrawn substantially according to FIG. 14, described below.

The deposit-receiving region of the substrate layer is in asurface-treated state for enhanced adhesion of deposits of biologicalmaterial thereon, preferably the surface treatment being that providedby a corona treater.

At least one intervening layer lies between the rigid support and theultra-thin polymer substrate layer, the intervening layer adherentlyjoined on each of its oppositely directed faces to substance of thedevice, e.g. immediately adjacent materials on opposite sides of anintervening layer are not as adhesively compatible with each other aseach is with the intervening layer.

The intervening layer is an adherent oxide of metal, preferably an oxideof tantalum or aluminum, or a silica based material, e.g. colloidalsilica or a soluble silicate such as sodium silicate.

The intervening layer is of substance selected from the group consistingof silane, epoxy silane, polylisine, PEI, GAP, adherent metal oxides,colloidal silica and soluble silicates.

A straight support surface e.g., planar or cylindrical is provided, andthe intervening layer is a drawn coating applied directly or indirectlyto the rigid member in the direction of the straight surface, preferablydrawn substantially according to FIG. 13, described below, preferablythe intervening layer comprising colloidal silica or a soluble silicate.

A surface of one of the constituents of the device, prior to beingunited with a next constituent layer of the device, is in asurface-treated state for enhanced adhesion of that surface to the nextconstituent layer.

An intervening layer is at least partially opaque, the intervening layerblocking at least 30%, preferably blocking at least 50%, or oftenpreferably blocking at least 70% of incident radiation at a wave lengthcorresponding to the stimulating or emission wavelength of a fluorophoreor luminescent tag on biological material.

The rigid support has characteristic fluorescence or luminescence inresponse to incident stimulating radiation, an intervening layer beingeffective to at least substantially limit penetration of incidentstimulating radiation from the substrate layer to the support or limitpenetration of fluorescent or luminescent radiation from the support tothe substrate layer, or both.

The intervening layer is electrically conductive and an electricterminal may be associated with the intermediate layer for applying avoltage potential to the layer to promote binding or rejection ofbiological material exposed to the substrate layer.

The device may be constructed and arranged to support biologicalmaterial for microscopy, and an intervening layer of an oxide of metal,preferably oxide of tantalum or aluminum, may be adapted to serve atleast one of the functions, preferably more than one, of adhesivelyuniting, either directly or indirectly, the rigid support with thedeposit-receiving substrate layer, of providing an opaque barrier toprevent or substantially limit light passing between thedeposit-receiving substrate layer and the support, of providing aradiation-absorptive layer to heat the substrate layer, or of providingan electrically conductive layer as a means to heat,.electricallycharge, inspect, treat or excite the substrate layer.

The substrate layer is adapted to receive a deposit of biologicalmaterial and to be temporarily engaged by an object adjacent thedeposit, as by an elastomeric gasket, wherein the substrate layer isinterrupted so that adherence of the substrate to the object does notdisrupt the array when removing the object, preferably an interveningadhesion promoting layer beneath the substrate layer being interruptedsuch that a substrate layer applied thereto is disrupted, for example bya moat, formed by a gap in a pattern of a metal oxide adhesion promotingintervening layer, in some cases the substrate layer being applied as acontinuous fluid coating which separates on drying at interruptions ofan adhesion promoting layer, such as the metal oxide layer.

For use in microscopy, an outer surface of the substrate layer isconstructed to receive deposits of biological material thereon inposition exposed for direct illumination and inspection from theexterior.

In some cases, devices according to the invention are functionally atleast partially transparent to pass effective light in at least onedirection between a deposit on the substrate layer and through the rigidsupport, as in the case of multiwell plates. For instance the device maybe arranged to enable illumination of a deposit of biological materialon the substrate layer via the rigid support, or the device may bearranged to enable microscopic inspection of a deposit of biologicalmaterial on the substrate layer via the rigid support, or the device maybe arranged to enable microscopic inspection from both the exterior sideof the substrate layer side and via the rigid support.

According to another aspect of the invention, a device for immobilizingbiological material is provided comprising a polymer substrate layerhaving biological immobilizing properties, preferably for protein ornucleic acid, the substrate layer deposited on a rigid support, andhaving an outer deposit-receiving region exposed to receive biologicalmaterial, wherein the rigid support defines a straight support surfacee.g., planar or cylindrical, and the substrate layer is a drawn coatingapplied directly or indirectly to the rigid member in the direction ofthe straight surface, preferably drawn substantially according to FIG.14 to be described below, preferably there being at least oneintervening layer, which lies between the rigid support and theultra-thin polymer substrate layer, the intervening layer adherentlyjoined on each of its oppositely directed faces to substance of thedevice and preferably used where immediately adjacent materials onopposite sides of the intervening layer are not as adhesively compatiblewith each other as each is with the intervening layer.

According to another aspect of the invention, a device for immobilizingbiological material is provided comprising a polymer substrate layerhaving biological immobilizing properties, preferably for protein ornucleic acid, the substrate layer deposited on a rigid support, andhaving an outer deposit-receiving region exposed to receive biologicalmaterial, wherein at least one intervening layer lies between the rigidsupport and the polymer substrate layer, the intervening layeradherently joined to substance of the device on each of its oppositelydirected faces, and the intervening layer is at least partially opaqueto radiation employed to stimulate emission from the biologicalmaterial, the layer limiting or preventing transmission of radiationfrom the rigid support, used preferably in cases in which immediatelyadjacent materials on opposite sides of the intervening layer are not asadhesively compatible with each other as each is with the interveninglayer.

According to another aspect of the invention, a device for immobilizingbiological material is provided comprising a polymer substrate layerhaving biological immobilizing properties, preferably for protein ornucleic acid, the substrate layer deposited on a rigid support, andhaving an outer deposit-receiving region exposed to receive biologicalmaterial, wherein at least one intervening layer lies between the rigidsupport and the polymer substrate layer, the intervening layeradherently joined to substance of the device on each of its oppositelydirected faces, preferably used where immediately adjacent materials onopposite sides of the intervening layer are not as adhesively compatiblewith each other as each is with the intervening layer, and wherein anintervening layer comprises an electrically conductive layer, forinstance, the electrically conductive layer being associated with atleast one electrical terminal and the conductive layer and theelectrical terminal are constructed and arranged to provide a voltagepotential to the receiving surface of the device to promote binding orrejection of material exposed to the outer deposit-receiving surface ofthe substrate layer.

In such cases involving intervening layers, preferably one or morelayers of the device is in a surface-treated state for enhanced adhesionto an overlying layer or for adhesion of deposits of biological materialthereon, for example the surface treatment being that provided by acorona treater.

One or more of the following novel features is useful with each of thevarious aspects of the invention that have been described.

The substrate layer is substantially solid, preferably having athickness less than about 5 micron, preferably less than 3, 2 or 1micron and in preferred embodiments between about 0.1 and 0.5 micron.

The substrate layer, at least in its outer region, is micro-porous,preferably the substrate layer being micro-porous throughout itsthickness, and preferably, the substrate layer having a thickness lessthan 3 micron, preferably less than 2 or 1 micron.

The substrate layer is nitrocellulose or polystyrene, preferablyresiding on an intervening surface adhesion promoter layer, preferablythat intervening layer comprising an adherent oxide of metal, preferablytantalum or aluminum oxide, or comprising colloidal silica or a solublesilicate that is preferably a drawn coating.

More broadly the substrate layer is selected from the group consistingof nitrocellulose, polystyrene, cellulose acetate, cellulose triacetate,ethyl cellulose, activated nylon, polytetrafluoroethylene (PTFE),polyvinyl difluoride (PVDF), polyamide, polyvinylchloride (PVC), divinylbenzene, and agarose.

A surface-treated state of the substrate of an intervening layer is theresult of exposure of the respective surface to corona or flametreatment, bombardment with charged particles including electrons, ions,and sub-atomic particles, or exposure to electromagnetic radiation, suchas ultraviolet, gamma, or X-ray wavelengths.

The rigid support is a microscope slide, preferably a frosted microscopeslide, preferably a blank frosted glass slide, preferably the microscopeslide being coated with a metal oxide, or has an adhered drawn coating,as by the process shown in FIG. 13, described below, or both, or thedevice is in the form of a bio-cassette, a CD disk, the bottom of amulti-well plate, or a hollow tube.

A multiwell plate comprises an upper well-defining structure and abottom plate comprising a substrate layer, the upper well-definingstructure and the upper surface of the bottom plate member being ofdissimilar material, and an adhesion promoting intervening layer beingdisposed between the upper well-defining structure and the bottom plate,the well-defining structure being polystyrene or similar polymer and thesupport of the plate member being glass, fused quartz, silicon orceramic, the adhesion promoting layer preferably comprising an adherentmetal oxide, e.g., tantalum oxide or aluminum oxide, and preferably alayer of polystyrene or similar compatible polymer being disposed overthe adhesion promoting layer to which the well-defining structure isbonded.

A localized reference deposit of stable fluorescent material is providedon the device, preferably characterized by a broad fluorescencespectrum, e.g. polyimide, and preferably disposed on the device inposition to be read by an optical instrument such as a microscope or CCDsensor for quality control of production of the device, or as anintensity calibrator during reading of fluorescence of substancedeposited on the substrate layer.

The support is selected from the group consisting of glass, fusedquartz, silicon, plastic, PMMA or polystyrene, or where not requiringtransparency, of ceramic or a metal such as gold, aluminum or silver.

The outer surface of the substrate layer is generally flat and arrangedto receive the deposit of a spotted array of bio-material, or alreadycarries an array of biomaterial spots either in unreacted state or in areacted state as a result of performance of an assay in which at leastsome of the said spots carry a fluorescent label.

According to another aspect of the invention, a method is provided offorming the device including applying directly or indirectly to therigid support a fluid containing the polymer of the substrate layerunder conditions to form the substrate layer, preferably by drawing therigid support from a bath of coating composition, preferably thisincluding applying an adhesion-promoting layer directly or indirectly tothe rigid support before application of the substrate layer, preferablyby applying a metal, preferably tantalum or aluminum and allowing it tooxidize, or applying soluble silica or colloidal silicate by drawingfrom a bath. In these cases, preferably forming conditions aremaintained to produce a solid film coating as the substrate layer, orare maintained to produce a micro-porous substrate layer.

The methods preferably include post-treating the substrate layer coatingto alter its structure or properties, for instance subjecting thecoating to corona discharge or to reactive gas or to structure-changingradiation, or to a combination of solvents selected to form pores.

The substrate layer is formed of nitrocellulose or polystyrene.

An opaque layer is applied directly or indirectly to the rigid supportbefore applying the substrate layer, for instance a metal oxide layer isapplied of sufficient thickness as to be at least partially opaque, toserve as a barrier to transmission of radiation.

One or more surfaces is treated during forming of the device byincreasing surface energy, or altering the surface structure, oraffecting biological binding affinity in the case of a receiving surfacefor a molecule or bio-material of interest or of a material wished to berejected, for instance subjecting the surface to corona or flametreatment, or bombardment with ions or sub-atomic particles, or exposingthe surface to selected electromagnetic radiation such as gammaradiation or X-rays.

In a commercial manufacturing process for production of a support filmfor spotted biological specimens for reaction or analysis andfluorescence measurements for quality assurance, the step of measuringfluorescence in response to excitation of the support with a wavelengthintended to be used with the biological specimen, preferably the processparameters being selected to produce a coating having a fluorescencelevel of no more than about five times, preferably no more than aboutthree times, or two times the fluorescence obtained from the uncoatedrigid member.

For producing a substrate support for spotted biological specimens forreaction or analysis, performing the steps comprising: (a) at leastpartially immersing a rigid member that defines a straight supportsurface (e.g., planar or cylindrical) in a vat containing a coatingsolution comprising a biologically compatible organic film-formingcomposition which includes at least one volatile solvent, (b)progressively drawing the member from the solution along a fixed pathinto a still environment, (c) the fixed path being generally parallel tothe straight support surface, (d) the conditions of the stillenvironment enabling the solvent to evaporate to leave a drawn coatingof the composition adhered to the support surface, for example the rigidmember being a microscope slide, or a component of a bio-cassette, orthe bottom member of a multiwell plate and the conditions beingmaintained to cause the formation of a solid film, e.g. a thin film ofpolystyrene or nitrocellulose, for example the film-forming compositioncomprising nitrocellulose dissolved in amyl acetate or an organicsolvent such as acetone, dimethylsulfoxide, ethyl acetate, or othercommon organic solvents or the organic film-forming compositioncomprising polystyrene dissolved in an organic solvent such as acetone,dimethylsulfoxide, ethyl acetate, or other common organic solvents, orthe method being conducted to produce a micro-porous membrane, such as anitrocellulose micro-porous membrane, as by dissolving nitrocellulose inmethyl acetate, ethyl alcohol, butyl alcohol, water, and glycerol.

Providing the support in the form of a disc having an adherent surfacefor a substrate coating fluid, spinning the disc individually,preferably the disc being in the form of a compact disc (CD), andproviding a substrate coating fluid in the center region of the discwhile it is spinning, to enable radial distribution of the fluid andremoval of all except that retainable by action of surface forces.

Another aspect of the invention is a method of conducting an assayincluding providing a device according to any of the above descriptionor by the methods of the above description, applying an array of spotsof bio-material to the substrate, conducting an assay which tags atleast some of the spots with a fluorescent or luminescent label, and,after washing the array, reading the array by fluorescent or luminescentdetection, preferably the assay being based on protein-proteininteraction, or involving an array comprising nucleic acid or othergenetic material, or comprising viruses, peptides, antibodies,receptors, cDNA clones, DNA probes, oligonucleotides including syntheticoligonucleotides, or polymerase chain reaction (PCR) products, or thearray comprising plant, animal, human, fungal or bacterial cells, ormalignant cells, or cells from biopsy tissue. Preferably the reading isaccomplished with a CCD sensor, preferably accomplished by the darkfield reflection mode.

DESCRIPTION OF DRAWINGS

FIGS. 1, 1A and 1B, in three stages of increasing magnification, arediagrammatic cross sections of a porous polymeric membrane of the priorart showing a possible distribution of biological material relative tothe volume of the porous structure of the membrane.

FIGS. 2 and 2A are, in two stages of increasing magnification, crosssections, similar in type, respectively, to FIGS. 1 and 1A, of anultra-thin micro-porous bio-material-immobilizing polymer membraneembodiment of the invention, showing a possible distribution ofbiological material within the porous ultra-thin coating.

FIGS. 3 and 3A are views similar to FIGS. 2 and 2A, respectively, of anultra-thin solid bio-polymer-immobilizing polymer membrane.

FIG. 4 is a diagram illustrating vacuum deposition of metal onto amicroscope slide.

FIG. 5 is a general work flow diagram of manufacturing thin film coatedmicroscope slides.

FIG. 6 shows a typical sequence of operations to coat slides. Dashedlines represent alternative steps.

FIGS. 7A and 7B show two frosted microscope slides; the frosted slide in7B bears silk screen printing over the frosted area and over a thinborder region surrounding the spotted array-receiving area.

FIG. 8 diagrammatically illustrates coating a slide with tantalum from asource in a vacuum chamber, to be followed by oxide formation.

FIG. 9 illustrates diagrammatically laser ablation of a microscope slideto produce a marking on its surface, while FIG. 9A is a plan view of theresultant slide.

FIG. 10 depicts a well of a microwell plate containing a fluorescentcalibration composition in which a pin is dipped to receive thecomposition for spotting.

FIG. 11 shows diagrammatically a spotted microarray of biological spotsamong which is a pattern of fluorescent calibration spots produced withthe composition of FIG. 10.

FIG. 12 shows diagrammatically a microscope slide undergoing coronatreatment as it is translated.

FIG. 13 shows a coating station at which a drawn film of colloidalsilica or soluble silicate is applied to a glass microscope slide.

FIG. 14, similar to FIG. 13, shows a substrate coating station in whichthe tank holds a composition for producing a drawn film or membranesubstrate layer on a microscope slide.

FIG. 15 illustrates microscope slides being withdrawn from a polymercomposition for applying a drawn ultra-thin substrate film while FIG. 16illustrates the dried substrate; FIGS. 17 and 18 similarly illustrateforming a drawn microporous membrane. Note that the microscope slidethickness in each figure has been broken away to suggest the relativelysmall thickness of the applied coatings.

FIG. 19 illustrates diagrammatically spotting an array of biologicalmaterial upon an ultra-thin bio-material-immobilizing polymericsubstrate layer, produced as above, to be followed by performance of anassay that binds and fluorescently tags selected spots in the array,followed by washing and imaging.

FIG. 20 illustrates fluorescent imaging, with a CCD sensor, by darkfield reflection mode, of the spotted array resultant from the spottingof FIG. 19, employing a porous ultra-thin, immobilizing polymersubstrate, after the array has been altered by an assay technique andsuitably washed.

FIG. 20A illustrates imaging a spotted array on a solid ultra-thinbio-material-immobilizing polymer substrate, employing a confocalepi-microscope.

FIG. 21 is a diagrammatic perspective view of a bio-cassette having aprotein microarray on an ultra-thin membrane, e.g., for supply toclinical laboratories for patient diagnosis, while FIG. 22 is amagnified view of a portion of FIG. 21 showing, in addition tobiological spots, fluorescent intensity calibration and fiducialcalibration spots.

FIG. 23 is a diagrammatic plan view, on an enlarged scale, of an arrayof protein spots on a substrate layer, surrounded by a moat in thesubstrate layer, while FIG. 24A is a diagrammatic plan view of a portionof a microscope slide showing four such micro arrays, FIG. 24B is adiagrammatic transverse cross-sectional view taken on line 24B-24B ofFIG. 24A, and FIG. 24C is a highly magnified view of a portion of PIG24B.

FIG. 24D is a view similar to FIG. 24A with a gasket in place and FIG.24E is a similar view with the gasket removed and by a circle, showingthe field of view of a microscope inspecting the slide.

FIG. 25 is a diagrammatic perspective view of a “CD” shaped bio-cassettewith cover removed;

FIG. 25A is a diagrammatic perspective view of a “CD” spinner,illustrating spin coating of a single “CD”-shaped support; and FIG. 25Bis a block diagram of a forming sequence for forming a “CD” rigidsupport having an ultra-thin substrate coating of abio-polymer-immobilizing polymer.

FIG. 26 illustrates the application of an electrical potential to asubstrate and an effect that can be obtained.

FIG. 27 is a perspective view of an assembled multi-well-plate, FIG 28is an exploded view of the multi-well plate of FIG. 27, FIG. 29 is avertical cross-sectional view of the assembly of FIG. 27, and FIG. 30 isa magnified cross-sectional view of the bottom plate of the assembly ofFIGS. 28 and 29.

FIG. 31 is a view similar to FIG. 30 of an alternative constructionwhile FIGS. 32 and 33 are cross-sectional views of two parts of amulti-well plate prior to assembly of another construction while FIG. 34is a similar view of the completed assembly and FIG. 34A is a view atincreased magnification of a portion of the cross-section of FIG. 34.

Like reference symbols in the various drawings indicate like elements.All percentages given in the formulations are by weight.

DETAILED DESCRIPTION

Referring to prior art FIG. 1, a conventional membrane 10 ofnitrocellulose of thickness t_(m) of e.g. 12 to 15 micron isdiagrammatically depicted upon the surface of a transparent, rigidsupport 12, e.g. a glass microscope slide of thickness t_(s) of e.g. 1mm.

As suggested in the highly magnified diagram of FIG. 1A, thenitrocellulose layer of thickness t_(m) is micro-porous in nature. Intoits interstices, to a limited depth t_(b), biological material B of adeposit is shown to have migrated as the result of a spotting or otherdeposit technique. Typically the biological material B, deposited in aliquid suspension, migrates downwardly as the liquid is absorbed orprogresses through the thickness of the membrane 10. The molecules ofbiological material bind to sites on the elements 10 a of the substrate,see FIG. 1B, and are immobilized, while the carrier liquid disperses andevaporates. The depth of penetration t_(b) of significant concentrationsof biological material B depends upon factors such as degree of porosityof the nitrocellulose or other immobilizing membrane, size andconfiguration of the specific bio-material molecules in the suspension,overall viscosity of the liquid suspension, and method of deposit Inmany spotted arrays of the prior art, depth of penetration t_(b) ofmolecules B is substantially less than the overall thickness t_(m) ofthe membrane, typically substantially less than half. For instance, witha conventional nitrocellulose membrane of thickness t_(m) of 12 to 15micron, the depth of penetration t_(b) of significant quantities of thebiological molecules is often of the order of one third or less than thethickness t_(m), e.g. t_(b) is equal to 3 or 4 micron, thoughpenetrations up to 6 or 7 micron at other times may be observed.

As depicted in FIG. 1B, immobilized molecules B₁ reside on the surfaceof a constituent element 10 a of the micro-porous membrane. During assayof the spotted array, a fluorescently or luminenscently tagged moleculeof reagent R_(t) binds to molecule B₁. When excited by stimulatingradiation SR to fluoresce or luminescence, it emits radiation F, of wavelength characteristic of the tag.

Referring to the embodiment of FIGS. 2 and 2A of the present invention,an ultra-thin micro-porous immobilizing membrane 20, of thicknesst_(ut), e.g., of e.g. 2 to 3 micron, is coated upon the support 12,which again may be a glass microscope slide. Adhesion of the ultra-thinlayer is enhanced by an intervening adhesion-promoting layer 14. Layer14 may be, in the case of a glass support, silane or epoxy-silane (whichoffers a covalent bond to nitrocellulose and consequently is able tosupport a mono-layer of nitrocellulose molecules), or other commonsurface adhesion promoters such as PEI (Polyethylene imine), and GAP(gamma amino propylene).

Preferably, however a layer of soluble silicate such as sodium silicateor colloidal silica, or a thin oxide of metal layer, such as of tantalumoxide or aluminum oxide, is employed or in some preferred cases,successive layers of one from each of these groups is employed.

As depicted in FIG. 2A, as a result of deposit by spotting, significantquantities of the biological material B penetrate to a depth t_(b) equalto the majority of the thickness of the immobilizing membrane 20,preferably to more than two thirds of the thickness, or, as depicted inFIG. 2A, substantially through all of the 2 to 3 micron thickness t_(ut)of the ultra-thin micro-porous membrane.

Comparison of prior art FIGS. 1 and 1A with the embodiment of FIGS. 2and 2A, illustrates advantages of the invention. For the comparison, weassume that the specific density and microscopic structure of the priorart membrane 10 of thickness t_(m) of, say 12 micron, is the same as themicroscopic structure of the ultra-thin membrane 20 of thickness t_(ut)of FIGS. 2 and 2A. Also, we assume for purposes of discussion, apenetration depth t_(b) of 3 micron of the biological material in bothFIGS. 1A and FIG. 2A, with the same number of bio-molecules beingimmobilized in the same distribution in each case. According toprinciples already explained, the fluorescent signal attributable to thetagged molecules will be equal in the two cases, but, because the volumeof the nitrocellulose or other immobilizing substrate material, per unitarea of FIG. 1A, is about four times greater than the volume per unitarea of FIG. 2A, the self-fluorescent noise signal from thenitrocellulose or other immobilizing material will be significantlygreater with the conventional prior art membrane of FIG. 1A. Even if thesubtraction correction is employed, such correction is never perfect,the error generally being proportional to the size of the original errorsignal. Thus, improvement in corrected signal is obtainable with theembodiment of FIGS. 2 and 2A.

Other factors may result in even greater improvement by use of theultra-thin layer. Assume a suspension of biological material in a spotquantity has a tendency to penetrate deeper than the depth t_(b) of 3micron depicted in FIG. 1A. In the case of FIG. 1A, some of themolecules penetrate deeper, and those deeper molecules receive lessexcitation due to their greater depth and suffer loss of theirfluorescent signal as the radiation makes its way back to the surfacethrough the diffusive porous medium. This detrimentally affects thesignal-to-noise ratio.

The continuous ultra-thin membrane of the embodiment of FIG. 2 and 2A isfound to retain a key attribute of conventional much thickernitrocellulose membranes of the prior art, that of enabling imaging theunoccupied membrane between spots to enable subtraction of a valuerepresenting detrimental background fluorescence. Therefore, it is seenthat the embodiment of FIGS. 2 and 2A has significant advantages overthe spotted mixture approach recently proposed by Pinkel and by Audeh,et al., referred to earlier.

Referring now to FIGS. 3 and 3A, in this embodiment a solid ultra-thinfilm of immobilizing substrate material 20′ is formed by the techniquesmentioned Upon an adhesion promoting layer 14 on the support 12, anultra-thin solid coating 20′ of nitrocellulose or otherprotein-immobilizing polymer substrate material is formed of a thicknesst_(uts) of e.g. 0.1 to 0.5 micron. The resultant membrane, being solid,i.e., substantially non porous, as well as being continuous in X, Yextent, presents a superficially planar array-receiving surface (albeit,as with any surface, the surface exhibits microscopic or submicroscopicroughness over which the binding sites of the material aredistributed.).

At a first order of approximation, all binding sites of this generallyplanar surface are exposed for binding of the biological material.Substantially no binding sites are in the shade of over-lyingimmobilizing material. For reasons explained above, this arrangementprovides more of what may be called “first order” binding sites, andhence permits highly efficient use of the biological material, should itbe in short supply, as well as efficient use of reagents that may beexpensive and in small quantity.

As with the embodiment of FIG. 2 and 2A, the embodiment of FIGS. 3 and3A, also permits subtraction of background signal, and hence canrepresent a significant advantage over the Pinkel and Audeh et al.,approach, as well as significant advantage over prior commerciallyavailable substrates.

It is important to note that the substance of the ultra-thin substratelayer in both FIGS. 2 and 3 is nitrocellulose suitable for immobilizingprotein molecules such as viruses, peptides, antibodies, receptors, andother proteins or to a wide range of biological materials including,plant, animal, human, fungal and bacteria cell, cDNA, DNA probes,oligonucleotides, polymerase chain reactions (PCR) products, andchemicals. An ultra-thin substrate layer of polystyrene, according tothe invention, is also particularly effective for such biologicalmaterials.

In forming the devices of FIGS. 2 and 3, prior to applying theultra-thin layer, as shown in FIG. 4, glass slides 12 are exposed tovapor deposit conditions or sputter coating conditions for applicationof a metal-based coating, e.g., tantalum, which oxidizes to formtantalum oxide, or aluminum, which oxidizes to form aluminum oxide.

The metal for the oxide coating applied e.g. by sputter coating can beof Angstrom range thickness. By a simple protocol of opticaltransmission measurements of resultant layers over a coating series ofchanged duration, the metallizing conditions are determined according tothe desired degree of transmissivity or opacity desired. Similartechniques are common in the design and production of optical coatings,to which reference is made. For present purposes, it has been shown thatcoatings 54 of tantalum, converted to tantalum oxide, that permit 90%transmission to almost no transmission have effective adhesion qualitiesfor instance, to glass, to serve as the support 12, as well as tonitrocellulose and polystyrene, desirable materials for thebio-compatible substrate 56. At 70% transmission (blocking 30%) or less,e.g. 50% (blocking 50%) or 30% transmission (blocking 70%), an angstromrange thickness layer is effective to reduce or substantially eliminatefrom sensing in reflective mode, any fluorescence, luminescence or strayradiation that may originate in or enter through the substance of aglass microscope slide or other transparent, rigid support.

After application of such an oxide layer, or other adhesion promotinglayer such as described below, or combination of such layers, the slidesare stored in a controlled dry environment at room temperature, readyfor the next step.

The coatings described above ensure proper adhesion of thenitrocellulose or other ultra-thin immobilizing substrate polymer to theglass support 12. A number of other adhesion promoters can also be used,sodium silicate or colloidal silica presently being preferred among thealternatives.

Also, microscope slides coated with other adhesion promoters may bepurchased from commercial houses such as Erie Scientific.

According to a feature of the invention, an advantageous method ofpreparing thin-film or microporous membrane coated slides employsdrawing slides from immersion in a polymeric solution. Generalized stepsfor the method are shown in FIG. 5. Slides are prepared prior toimmersion, are immersed and drawn from a polymeric solution, which isthen followed in many advantageous cases by a post drawing surfacetreatment such as exposure to corona discharge, etc. As previouslynoted, the resultant coating is referred to herein as a “drawn” coating.

The slide preparation can advantageously employ steps of applying one ormore layers of adherence promoting layers and, as well, may employsurface treating the surface of the base support or one or more of theadded layers to promote adhesion of the next layer.

FIG. 6 illustrates the succession of preferred steps (with preferredalternatives) in a method used to prepare commercial glass slides forprotein or nucleic acid processing. Alternative steps are indicated bydashed line in FIG. 6. For example, after the initial wash and cleanstep, a slide is silk screened or coated with tantalum oxide, inpreparation for marking and serialization.

Referring to FIGS. 7A and 7B, a silk-screened pattern 104 is applied. Itcovers the frosted side 102 of the slide 100 as well as providing anyframe 106 that may be desired. A frame, tracing the outer periphery ofthe slide, may have a width of 1 to 3 mm (preferably 2 mm). The regionover the frosted end 102 may be marked and serialized e.g. with acommercial laser marker. The peripheral frame may serve both forprotection and aesthetic purposes.

Referring to FIG. 8, alternatively, or in addition, a uniform metalcoating 112, e.g. of tantalum oxide, is applied by sputtering or vapordepositing atoms from a source 108 onto the slide 100 in a vacuumchamber 110. This is followed by air oxidation. The oxide coating thusprepared is preferably at least partially opaque and employed to permitlaser marking and serialization, as well as serving as an efficientadhesion promoter. Laser ablation may also serve advantageously toseparate the region to be spotted into a number of separate sub-regions.The ablation may be performed on a coated substrate, or performed on anadherence promoting layer that leads to segmentation of a fluid-appliedlayer, as later described.

In important cases, using a transparent material for the rigid support,an opaque coating either the metal oxide, or other opaque layer, appliede.g. by printing techniques, blocks light transmission between thesupport and the bio-material carrying substrate layer.

In embodiments when a transparent slide is desired, an oxide coating 112is omitted or applied very thinly and the object is laser-marked at thesilk-screen pattern 104.

FIG. 9 illustrates use of laser ablation 113 to mark the coating 112over the frosted region 102 to serialize the slides 100 or addidentification or registration markings 116 for automatic optical unitor information retrieval. Markings produced by laser ablation 113 may beused to enhance data acquisition reliability using various devicesincluding, advantageously, commercial bar code readers.

After application of an adhesion promoting layer 112, one ore moredurable sensitivity and geometry calibration spots may be locallydeposited on slide 100. In a distribution such as suggested in FIGS. 11and 23 reference spots may also be applied to the exterior surface ofthe substrate layer 20 or 20′, here illustrated as in a patterndistributed across the array.

Reference spots, such as those at the corners of the array, may beemployed as fiducial markings for geometrical reference, as by theimaging device. By suitable choice of materials such as the polyimidepolymer mentioned, the same reference spots can serve as standards forthe reading equipment to determine and accommodate long term variationsin the optical instrumentation, such as light intensity or detectorsensitivity. Likewise, they can be used to compensate for variation inillumination level over the area, e.g. to normalize the data acrossareas illuminated at different intensities.

Temporally stable spotting material such as polyimide (Kapton) may beused as the calibration spots, deposited in solution in a solvent usinga commercially available spot printer. Preferably, the calibrationcompound is selected to have a broad fluorescence spectrum. These usesof reference spots are discussed further below with reference to thereader of FIG. 20.

Calibration markings 420 applied at the slide-manufacturing site, eitherbelow or on top of the substrate layer, can be employed in qualityassurance steps whereby fluorescence of the ultra-thin film coatedslides is measured following the final surface treatment step asindicated in FIG. 6. For quality assurance, fluorescence maybe measuredfrom each unit or on a statistical sampling basis in accordance withquality control protocols.

Following the above steps, the slides are subsequently washed and, asshown in FIG. 12, exposed to surface treatment to promote adhesionproperties of the film or membrane. During corona treatment, the slide100 is translated under a jet of reactive species 124, e.g. ozone,produced by a corona treater 122 held at a distance d. Ifelectromagnetic radiation such as ultraviolet light is utilized, asuitable source of photons is used to process the surface, e.g. a UVlaser beam may be moved across the surface in suitable raster scan.

Referring to FIG. 13, immersion and drawing from a bath is employed forapplying a layer to promote adhesion of the final substrate layer to belater applied. Drawing is preferably performed in a clean laboratoryenvironment in a comfort humidity and temperature zone, preferably 33%humidity and 26° C. The cleaned and preferably recently (less than oneday) corona treated slides 302 are immersed in a tank 300 of solublesilicate or colloidal silica, preferably a 3.3% solution of LUDOX CLfrom Sigma-Aldridge Co., and drawn out in the direction of the plane ofthe slide at a steady rate of approximately 0.5 in/min. The result is acoating of uniform thickness that serves as an adhesion promoting layer.(When not in use, the tank 300 should be closed to avoid evaporation,and the liquid should be stirred a minimum of once per day.)

Slides not coated with metal oxide preferably receive such asilica-based coating. Slides with a metal oxide deposit may or may notreceive such a coating.

In preferred embodiments, a number of slides 302 are suspended from arack and processed simultaneously to apply the silica-based coating. Thegroup of slides attached to a carriage 306, such as a one-axis,vibration-free carriage linear-transport from Sherline Products inVista, Calif., is translated, from the bath preferably at a uniform rateand between preset positions in a controlled manner. The slides thuscoated with colloidal silica or silicate are air dried prior to beingcoated with the substrate layer material. The silica or silicate layerthus applied may be surface treated, e.g. according to FIG. 12, prior toapplication of the substrate layer.

FIG. 14 shows a preferred embodiment of a coating station forapplication of the film or membrane substrate that is to receive thespotted array of biological material. The station is similar inconstruction and operation to the mechanism of FIG. 13. The process isperformed in a still environment (i.e., substantially no air currents orvibrations), in vibration-free conditions, with control parameters oftemperature, humidity, and draw rate selected in accordance with thepurposes of the composition and the desired coating thickness.

Slides are immersed, then drawn from the bath as indicated in FIG. 14.FIGS. 15-18 diagrammatically illustrate aspects of the drawing processas well as post-drawing features. Slides 900 are shown edgewise alongtheir thinnest dimension t (typically about 1 mm for standard glassslides). FIGS. 15 and 17 represent drawing an immersed slide 900respectively from a thin film coating composition 902 and a microporousmembrane coating composition 904. During immersion, approximately ¾ ofthe length of a slide 900 is immersed in the polymer composition 902 or904, i.e. the portion intended to receive the spotted array. As theslides 900 are drawn in translation direction represented by arrow 906,the polymeric compositions 902 and 904 adhere to the slides and formcoatings 908 and 910 (stylized in the figures) on the surface of theslides 900.

As the slides are drawn and coated, the newly formed coating composed ofpolymer and solvent begins to dry. The initial thickness of the wetcoating is shown diagrammatically in FIGS. 15 and 17, comprising the sumof the resultant film thickness, t_(f) or t_(mm), and thickness t_(v)attributable to the volatile solvent. As solvent evaporates, the coatingremaining on the slide surface becomes thinner and upon completeevaporation, t_(f) is much thinner than t_(mm) by several orders ofmagnitude, indicated diagrammatically in FIGS. 17 and 18.

Note the slide 900 is shown broken away in FIGS. 15-18 to emphasize therelative thinness of the film and microporous membrane structures.

In the case of interruption of the adherence promoting layers ortreatment, the dried coating may form lines of discontinuities orseparation moats for purposes to be described later in reference toFIGS. 23 and 24A-E, see especially moat M in FIG. 24C.

In these embodiments, following drawing and drying of the polymercoatings, the slides are subjected to surface modification with coronatreatment (see FIG. 12) by translating the slide at a speed between 2and 8 cm/min (preferably 4.4 cm/min) while maintaining the exposedsurface to be treated normal and at a distance of between about 1 and 4cm, (preferably 2 cm ) from the jet of a standard 2.5 cm. round head ofa corona treater, e.g. laboratory treater model BD-20AC fromElectro-Technic Products Inc., of Chicago, Ill. operating near itsoptimal level.

The slides are transported between the various stations described by anoperator or by suitable robots e.g. as part of standard commercialequipment or as part of a continuous production line.

Preferably, the coating process, including drawing theadherence-promoting layer and drawing the substrate film or membrane, aswell as the post-drawing treatment, are performed in a clean room orwithin a hood.

The thus-formed substrate layers may also be subjected to other postforming treatments. For instance they may be treated tosubstrate-altering conditions, e.g. scanned under a laser or electronbeam selected to provide porosity or otherwise to alter the overallbio-material binding characteristics or other structural features of thepolymer membrane, such as for pore-forming with a laser or by animpinging fluid.

The slides, when completed, may be packaged as single units or inmultiples (such as 5 or 20 slides).

After this preparation, the slides are ready to be spotted to formmicroarray A as shown in FIGS. 11, 19, 22 and 23. For instance thetechniques explained in U.S. Pat. No. 6,269,846, which is herebyincorporated by reference in its entirety, may be used to form the arrayof spots 422. Simultaneously, according to a predetermined pattern,spots of reference material 420 may be strategically distributedthroughout the array as suggested by FIGS. 10, 11, 22 and 23. Followingthis, the array is subjected to assay conditions, such as are generallydescribed in the above-cited Chin et al., patent. After washing they areready to be imaged.

Referring to FIG. 20, imaging in a dark field reflectance mode may beaccomplished with a CCD sensor 124 positioned to view the array alongaxis A normal to the plane of the array via collection optics 127,spaced a distance h from the substrate. In this case the substrate layermay be microporous partially or throughout its depth or may be a solidfilm or a modified solid film. As shown, light for direct illuminationenters along an illumination axis A′, at an acute angle θ to the planeof the array. Distance h must be selected to enable such directillumination, with angle θ ranging between about 20° and 50°, here shownat 45°. Light L originates from a source 112 a, 112 b or 112 c ofwavelength selected to excite the fluorophore tag of the array, passesvia dichroic mirrors 156 b, 156 c to mirror 116 located to the side thatdirects the illumination along axis A′ at angle θ, onto thefluorophore-tagged array of spots resident on the ultra-thin substrate20 or 20′. The fluorescent emissions are collected by lens 127, througha selected filter 128A, B or C, thence through lens 126 to CCD camera124 under computer control 132. As before, the background subtractiontechnique is used with this system.

Referring to FIG. 20A, a confocal scanner imager 10 may also beemployed. Such a confocal microscope may be constructed, for instance,according to Minsky, U.S. Pat. No. 3,013,467, or a scanning confocalmicroscope may be employed, such as the Affymetrix '428 Scanner,mentioned above. Such imagers may be in accordance with U.S. Pat. Nos.6,185,030 and 6,335,824. The three patents of this paragraph are herebyincorporated by reference in their entireties.

For use in calibrating such imaging systems it is advantageous toprovide the distribution of reference spots of known fluorescence in thearray to be read.

With reference back to FIG. 10, as a step in forming the array of FIG.11 (as well as the arrays of FIGS. 22 and 23) pin 400, which may be oneof a set of simultaneously actuatable pins of a commercially availablespot printer, is dipped into reference composition 440 held, forexample, within one of the wells 442 of a microwell plate also beingused for printing the biomaterial array. Instead of bio-material, thisparticular well 400 holds a reference solution such as polyimide(Kapton) dissolved in a suitable solvent. Thus, a user-applied referencearray is applied when spotting the array of biological material. (Inother cases geometric reference spots or fiducials and intensitycalibration spots may be applied during manufacture of the device, e.g.before or after applying the substrate layer.)

FIGS. 10, 11 and 19 illustrate pin 400 contacting slide 100 to depositcomposition 440 forming spot S. Typical diameters for spots 422 ofbiological material are 150 micron and 300 micron, and the same sizespots may be used for the simultaneously-applied intensity calibrationreference spots 420, using the previously mentioned synthetic resinmaterial, such as a polyimide polymer (Kapton), which exhibitstemporally stable broad fluorescence properties. By being presented insolution with a suitable volatile solvent and deposited as spots on theslide surface, evaporation leaves adherent fluorescence intensitycalibration spots in a reference pattern interspersed with the morenumerous spots 420 of bio-material. The same printer used to deposit thearray 418 of biological material spots 422, may thus be used to depositcalibration spots 420 on the slide. The amount of material deposited forspots 420 is not critical since polymers such as Kapton are opticallyopaque, from which fluorescent emission 464 occurs at or near thesurface, with the reproducible quantum yields.

The use of the calibration spots 420, however applied to each slide,enables instrument self-calibration. In fact, auto calibration of theinstrument may be accomplished for each slide, using e.g. 6 calibrationspots on a slide.

It is further found that non-uniformities of illumination of amicroarray as may occur with the side lighting operating in dark fieldreflection mode can be accommodated with a dispersed pattern ofintensity calibration spots such as shown in FIG. 23 to normalize thedetected results.

FIG. 21 shows a pre-spotted bio-cassette (cover not shown), having aprotein-immobilizing substrate that may be offered to clinics, bearing adiagnostic protein array of spots 422 and intensity and/or registrationcalibration spots 420, see FIG. 22. The clinical laboratory may react apatient's fluid with the array to determine a disease condition.

Besides the planar rigid supports described, bio-immobilizing polymermay-be applied to other surfaces such as by chemical vapor depositionupon the bottoms of wells of multi-well plates or to inside surfaces ofhollow reaction tubes.

Referring to FIGS. 23 and 24A, a number of spotted micro arrays 418 areoften placed upon the same microscope slide 10, intended to be reactedwith different fluids, for different assays, for instance using extractsof blood from different subjects. To protect againstcross-contamination, typically a fluid-tight elastomeric gasket 430, inthe form of a grid of upstanding walls 432, is pressed tightly againstthe face of the microscope slide. This defines individual wells atindividual arrays 418. Such an arrangement is shown in FIG. 24D, inwhich spotted arrays 418 are individually surrounded by walls 432 of thegrid-form gasket 430 that is pressed tightly against the substrate layer20″ on the microscope slide surface.

Different aliquots of fluid are then introduced to the respective wellsand reacted with the arrays of spots, following which the fluid may beextracted and the bottom of the wells washed.

For reading the arrays, the gasket is removed, and the microscope slideplaced beneath a microscope, its field of view suggested by the circlein FIG. 24E. In the gasket removal step, the gasket sometimes adheres tothe substrate layer more tenaciously than the substrate layer adheres toits support, and the substrate is lifted or disrupted. This can destroyreadability of the micro arrays or introduce possibilities of error intheir microscopic examination.

According to the invention, a substrate layer 20″, e.g. ofnitrocellulose or polystyrene, is applied to a rigid support 12, as inthe previous embodiments with an intervening adhesion promoting layer14′ being first applied to the rigid support, but in this instance, apattern of disruptions or moats M is formed in the substrate layer 20″,such that each portion of substrate on which an array is located isisolated from the portions of the substrate layer upon which the gasketwalls press. It is found that a substrate layer so formed prevents themicro arrays from being disrupted when the gasket is removed.

This segmentation may be achieved for instance by laser cutting orotherwise scoring a preformed continuous substrate layer to provideseparation of the substrate of each array from adjoining substrate. In apresently preferred form, however, the moats M are formed duringformation of the substrate by a simple and inexpensive technique. Theadhesion promoting layer is applied with a pattern of interruptionscorresponding to the desired location of the moats M. Where the adhesionpromoting layer is a metal oxide layer, this may be accomplished bymetallizing the surface of the rigid support, as by sputter coating,through a grid-form mask, define lines in which no adhesion promoterreaches the rigid support, or by laser etching after metallization. Itis found that subsequent formation of the substrate layer by applicationof fluid, as by use of the drawing technique depicted in FIG. 14,produces the desired separation of the array portions of the substrate.Upon initial application of the fluid, the interruptions in the adhesionpromoting layer occurs are covered by fluid, such as nitrocellulose orpolystyrene in solution. But, as the fluid dries, the fluid is found torecede from the masked regions that have no surface adhesion promoter.This forms the desired isolating moats M in the substrate as shown inFIG. 24C. The moats may for instance have a width of 0.005 inch.

Referring to FIGS. 25, 25A and 25B, another system for producing anultra-thin bio-immobilizing product is provided. Round, thin “CD” typediscs 120 of relatively rigid material are formed at station A. Thisbase support structure may be of PMMA or polystyrene. Following this,using robots as developed in the industry, each automatically formeddisc is introduced at station B to a vacuum chamber to receive a thin,adhesion promoting coating 121 of metal such as tantalum or aluminum,such as has been applied to semiconductor discs in the semiconductorindustry. The coating is exposed to conditions forming an adherentoxide, e.g. tantalum oxide or aluminum oxide.

Following that, at station C, one-by-one, the automatically formed discs120 with adhesion promoting coating 121 are introduced to a spin coatingstation, C, formed of standard spinning equipment, e.g. from thesemi-conductor industry. A layer of coating solution is either appliedprior to introduction to each disc, or, as shown, is applied, first atslow rotation speed at the spin coating station, to the center of thedisc, the disc being supported and spun from beneath. Following this,the so-coated disc is spun at high speed, to remove from the surface ofthe disc all of the solution except that thin layer that is retained bythe inherent adhesion forces existing between the fluid and the metaloxide coating. Either while still at station C, or preferably at stationD, the applied layer is dried. At that station, or at a subsequentstation, the formed layer may be subjected to a desired post-treatment,and then may be assembled with cover and other structure to complete abio-cassette.

The FIG. 26 diagram of a composite is intended as a generalillustration, e.g. to illustrate the cross-section of a planarmicroscope slide, a bio-cassette, a disc-form substrate, the bottom of amicro-well plate or other useful configuration for presenting materialon a substrate for microscopic examination from the substrate side.

A support 12, e.g. of glass of 1 mm thickness, has a surface to which athin adherent metal oxide layer 54 has been applied, in the example,tantalum oxide, as before. To the outside surface of the adherent metaloxide layer 54 is applied a bio-compatible substrate 56, e.g. ofnitrocellulose or polystyrene, which may advantageously be of theultra-thin dimension described above or, depending upon imagingrequirements, may be thicker. Subsequently the bio-material spots areapplied.

In the embodiment of FIG. 26, novel advantage is taken of theelectrically conductive properties of the adherent oxide coating. Aterminal 70 is associated with conductive layer 54′″ underlying thesubstrate 56, and connected to a battery 72 or other controllablepotential source. As shown, a controller, 74, e.g. a switch andrheostat, has settings #1 and #2, typically more. As suggested in thisdiagram, at setting #2 a positive electrical potential is applied toconductive layer 54′″ of a level to increase the binding affinity of atarget molecule as well as an accompanying molecule of lesser charge.After binding from the parent medium, the potential level may be changedto lower level, not shown, as by use of the rheostat. The targetmolecule remains bound, but binding force is so reduced or eliminatedwith respect to the accompanying molecule that it is washed away orotherwise detached from the substrate. The potential may be changed frompositive to negative, setting #1, for use in situations where a selectednegative potential will repel undesired molecules while still retainingthe target molecule.

Referring to FIGS. 27-34 a, a multiwell plate 90 is comprised of abottomless upper structure 91 and a planar bottom glass plate member 95.The upper structure 91 is of extruded form, e.g., of polystyrene. Withinthe periphery of structure 91, an array of wells 92 is formed by a nestof tubular walls 94′. Other such honeycomb-type structures can beemployed.

As is well known in biotechnology, wells of multiwell plates areintended to receive aliquots of unknowns and reagents, and fluorophoretags are excited and read by transmission of excitation light from anobjective to and transmission of fluorescent radiation from the wells,across the transparent bottom plate, to an objective of a detectionsystem.

In the field, there has been difficulty in bonding such polystyreneupper structure to glass. In accordance with another aspect of thepresent invention, on the bottom plate of glass (or a plate of polymermaterial such as polycarbonate also adhesively incompatible withpolystyrene), a layer of adherence-promoting material is applied. In oneembodiment the layer is formed in a pattern which matches the pattern ofthe lower edges 94 a of the bottom-less upper structure 94 of themicro-well plate to be assembled. In the embodiment shown in FIGS. 27-34a the layer is uniformly applied across the upper surface of bottomplate 95. In the case that metal oxide layer is employed as the layer,the oxide coating is selected to be so thin as not to substantiallyimpair light transmission qualities of the bottom plate. The portion ofthe applied layer beneath the lower edges facilitates the manufacture ofthe composite well-plate, as it enables the upper structure to be ofmaterial (e.g. polystyrene) that is adhesively incompatible with thebottom plate (e.g. glass).

Referring to FIGS. 30 and 31, such an adhesively incompatible supportplate 95, e.g. of glass for forming the bottom of a multi-well plate isuniformly coated with a film of an adhesion promoter 96, e.g. thetantalum oxide discussed, followed by application of a uniform upperfilm 97 of polystyrene or other substance that is adhesively compatiblewith the upper structure as well as suitable as a substrate for spots ofbio-material. The bottom-less upper elements of the micro-well plate maythus be of polystyrene and joined to a uniformly coated glass. The unionmay be enhanced by heat or by the temporary presence of a solvent suchas Amyl Acetate or the temporary presence of a solution of polystyrenein such a solvent. The glass surface, coated with a thin film ofpolystyrene, may have its surface adhesion enhanced as described above,e.g. by corona treatment, to serve as a biomaterial receiving substrateat the bottom of the wells.

The uniform film of polystyrene coated over the adhesion promoter on theglass plate, may be an ultra-thin layer as previously described, and itsareas within the grid of the multi-well plate may thus be suitable forreceiving deposit of bio-molecules as previously discussed. Thusmicroscopic reading of the wells through the transparent bottom of themicro-well plate can be enhanced.

In the embodiment of FIGS. 32, 33, 34 and 34 a, a pattern 98 of tantalumoxide or other suitable mutually adherent composition is applied to theplate 95′, matching the geometry of the grid 94. In the spaces betweenthose deposits a material is applied, e.g. a composite of an adhesivepromotion layer 56 a of any of those mentioned, and a bio-receivingsubstrate 54 a of any of those mentioned, see FIG. 34A. For instance thecomposite may be an adherent oxide layer on the top of which anitrocellulose or divinyl benzene substrate is applied.

For further disclosure concerning the topics of (1) employing thecharacteristics of ultra-thin substrate layers in dark fieldillumination and imaging on a solid state array of sensors of size oforder of magnitude of the array of spots, in general and in particularof nitrocellulose and polystyrene, and their methods of manufacture anduse, (2) metal oxide and other absorbent layers beneath the substratethat absorb excitation light serving to enhance the operation or makepractical a clinical fluorescence reader and (3) formation andutilization of intensity calibration marks in micro-arrays for servingto enhance the operation of a fluorescence reader, in particular oneusing a high intensity light emitting diode or diode array forexcitation illumination, reference is made to a further PCT applicationbeing filed simultaneously herewith, which likewise claims priority fromU.S. Provisional Ser. No. 60/476,512, filed Jun. 6, 2003.

Other features and advantages of the invention will be understood fromthe foregoing and the claims and are within the spirit and scope of theinvention.

1-50. (canceled)
 51. A device constructed for immobilizing material forexposure to liquid during an assay, the device comprising a polymerlayer on a rigid support, the layer having an outer deposit-receivingregion that has binding properties for the material, wherein the layercomprises nitrocellulose in the form of a coating of substantially solidfilm.
 52. The device of claim 51 in which the substantially solid filmis less than about 1 micron in thickness.
 53. The device of claim 51 inwhich the substantially solid film is between about 0.1 and 0.5 micronin thickness.
 54. The device of claim 51 in which the substantiallysolid film is a dried residue of a coating solution of nitrocelluloseand a volatile solvent.
 55. The device of claim 54 having an array ofspots of material applied to the substantially solid film for use in theassay.
 56. The device of claim 55 in which the array of spots ofmaterial comprises protein, peptides, antibodies, viruses, or nucleicacid or other genetic material, receptors, cDNA clones, DNA probes,oligonucleotides including synthetic oligonuceleotides, or polymerasechain reaction (PCR) products, or plant, animal, human, fungal orbacterial cells, or malignant cells or cells from biopsy tissue, orother bio-material.
 57. The device of claim 55 in which the spots arebetween about 100 and 400 micron in diameter.
 58. The device of claim 55in which the substantially solid film transmits light withoutsubstantial scatter.
 59. The device of claim 55 in which the rigidsupport is glass.
 60. The device of claim 55 in which the rigid supportis plastic.
 61. The device of claim 55 constructed to expose deposits ofthe material on the substantially solid film for direct illumination oroptical inspection from the exterior.
 62. The device of claim 61 inwhich a portion of the device below the substantially solid film issubstantially opaque to light from the solid film.
 63. The device ofclaim 62 in which the rigid support is substantially transparent and anadherent intervening layer between the solid film and the rigid supportis substantially opaque.
 64. The device of claim 55 in which deposits ofmaterial on the substantially solid film are subject to illumination orinspection via the rigid support, the device being functionallytransparent so that light can pass through the rigid support and thesubstantially solid film to the spots.
 65. The device of claim 64 inwhich there is at least one substantially transparent interveningadherent layer between the substantially solid film and the rigidsupport.
 66. The device of claim 51 in which the substantially solidfilm of nitrocellulose is adhered to the rigid support via one or moreadherent intervening layers.
 67. The device of claim 66 in which anintervening layer is comprised of an adherent metal oxide or asilicon-based material.
 68. The device of claim 66 in which anintervening layer is comprised of tantalum oxide or silane.
 69. Thedevice of claim 66 in which the rigid support is of glass and anadherent intervening layer is an adhesion-promoting layer comprised ofsilane, epoxy silane, polylisine, PEI, GAP, an adherent metal oxide,colloidal silica or a soluble silicate.
 70. The device of claim 51 inwhich the substantially solid film has its outer deposit-receivingsurface in treated state for enhanced immobilization of the material.71. The device of claim 70 in which the treated state is the result ofexposure of the surface to an energetic surface-altering condition. 72.The device of claim 71 in which the treated state is the result ofexposure of the surface to corona treatment.
 73. The device of claim 71in which the treated state is the result of exposure of the surface tocharged particles.
 74. The device of claim 71 in which the treated stateis the result of exposure of the surface to gamma radiation.
 75. Thedevice of claim 71 in which the treated state is the result of exposureof the outer surface to at least one of corona treatment, flametreatment, bombardment by charged particles comprising electrons, ionsor sub-atomic particles, or electromagnetic radiation of ultraviolet,gamma or X-ray wavelength.
 76. The device of claim 51 constructed toimmobilize material capable of being associated with a fluorophore tagfor optically-stimulated fluorescent emission analysis.
 77. The deviceof claim 55 combined with a reader for fluorescence from fluorescentlabels associated with at least some of the spots.
 78. The device ofclaim 77 in which the reader comprises a CCD sensor.
 79. The device ofclaim 51 in which the polymer layer is a drawn coating.
 80. The deviceof claim 51 in which the outer surface of the polymer layer is generallyflat, arranged to receive deposit of a spotted array of bio-material.81. A device for immobilizing biological material for analysis based onoptically-stimulated light emissions, the device comprising a polymersubstrate layer having biological immobilizing properties, the substratelayer deposited on a rigid support, and having an outerdeposit-receiving surface exposed to receive the biological material,wherein the substrate layer is comprised of nitrocellulose orpolystyrene that is ultra-thin, having a thickness t_(ut) less thanabout 5 micron, the substrate layer comprising a substantially solidfilm that is substantially transparent.
 82. The device of claim 81wherein said substrate layer has binding properties for protein ornucleic acid.
 83. The device of claim 81, wherein the device isconstructed to enable effective light to pass in at least one directionbetween a deposit on said substrate layer and through said rigidsupport.
 84. The device of claim 83, wherein said substrate layer isnitrocellulose.
 85. The device of claim 84, wherein the outer surface ofsaid substrate layer is generally flat and arranged to receive thedeposit of a spotted array of bio-material.
 86. The device of claim 81,wherein the deposit-receiving surface of said substantially solidsubstrate layer is in a treated state as the result of exposure of thesurface to an energetic surface altering treatment.
 87. The device ofclaim 81, wherein said support is selected from the group consisting ofglass, fused quartz, silicon, plastic, PMMA, polystyrene, ceramic or asolid metal.
 88. A method of forming the device of claims 84, 85 or 86including applying directly or indirectly to said rigid support a fluidcontaining said polymer of said substrate layer under conditions to formsaid substrate layer, preferably by drawing the rigid support from abath of coating composition.
 89. The method of claim 88 for devicesproduced with a solid film substrate layer, including maintainingforming conditions to produce a solid film coating as the substratelayer.
 90. The method of claim 89 including forming the substrate layerof nitrocellulose.
 91. The method of claim 90 for producing a substratesupport for spotted biological specimens for reaction or analysis, thesteps comprising: (a) at least partially immersing a rigid member thatdefines a straight support surface (e.g., planar or cylindrical) in avat containing a coating solution comprising a biologically compatibleorganic film-forming composition which includes at least one volatilesolvent, (b) progressively drawing the member from the solution along afixed path into a still environment, (c) the fixed path being generallyparallel to said straight support surface, (d) the conditions of saidstill environment enabling said solvent to evaporate to leave a drawncoating of said composition adhered to said support surface, for examplethe rigid member being a microscope slide, or a component of abio-cassette of the bottom member of a multiwell plate and theconditions being maintained to cause the formation of a solid thin filmof nitrocellulose, for example the organic film-forming compositioncomprising nitrocellulose dissolved in amyl acetate or an organicsolvent such as acetone, dimethylsulfoxide, ethyl acetate, or othercommon organic solvents.
 92. A method of conducting an assay includingproviding a device according to claim 85 applying an array of spots ofbio-material to the substrate, conducting an assay which tags at leastsome of the spots with a fluorescent label, and, after washing thearray, reading the array by fluorescent detection, preferably the assaybeing based on protein-protein interaction, or involving an arraycomprising nucleic acid or other genetic material, or comprisingviruses, peptides, antibodies, receptors, cDNA clones, DNA probes,oligonucleotides including synthetic oligonucleotides, or polymerasechain reaction (PCR) products, or the array comprising plant, animal,human, fungal or bacterial cells, or malignant cells, or cells frombiopsy tissue.
 93. The method of claim 92 wherein the reading isaccomplished with a CCD sensor.
 94. The device of claim 83 arranged toenable illumination of a deposit of biological material on saidsubstrate layer via said rigid support.
 95. The device of claim 83 or 94arranged to enable microscopic inspection of a deposit of biologicalmaterial on said substrate layer via said rigid support.
 96. The deviceof claim 85 in which an array of bio-material spots comprises depositson the deposit-receiving surface.
 97. The device of claim 96 in whichthe deposits are in unreacted state.
 98. The device of claim 96 in whichthe deposits are in a reacted state as a result of performance of anassay, at least some of the said spots being associated with afluorescent or luminescent label.
 99. The device of claim 81 wherein thedeposit-receiving surface of the substantially solid substrate is in atreated state as the result of exposure to corona treatment.
 100. Thedevice of claim 81 wherein the deposit-receiving surface of thesubstantially solid substrate is in a treated state as the result ofexposure to charged ions.
 101. The device of claim 81 wherein thedeposit-receiving surface of the substantially solid substrate is in atreated state as the result of exposure to gamma radiation.